The cells have been variously referred to as colony-forming fibroblasts, mesenchymal stem cells, or marrow stromal cells (MSCs), have attracted increasing interest both for their biological properties as multi-potential stem-like cells and their potential use for cell and gene therapy. And their potential use for cell and gene therapy. However, a major problem in the field has been that reproducible conditions for isolation and expansion of the cells in culture have not been defined. The overall aim of the present proposal is to develop effective conditions for the isolation and expansion of human hMSCs (hMSCs) in cultured based on three recent observations made in our laboratory: (a) We have developed culture conditions whereby hMSCs can be expanded over 10/8-fold without significant loss in replicative capacity. (b) We have found that replication of hMSCs in culture is regulated autocrine/paracrine factors that can be recovered from the culture medium. (c) We have identified a sub-population of small granular cells in cultures of hMSCs that are highly replicative and appear to be the earliest progenitors in the cultures. The Specific Aims of this proposal are: (1) Determine whether the highly replicative cells we have obtained after 10/8-fold expansion of hMSCs retain their multi- potentiality to differentiate into osteoblasts, chrondrocytes and adipocytes. (2) Isolate and characterize the secreted factors that regulate replication of hMSCs in culture. (3) Isolate the small, granular, and highly replicative cells we have identified in cultures of hMSCs and define their surface epitopes.